Urocortin-III and uses thereof

ABSTRACT

A search of the public human genome database identified a human EST, GenBank accession number AW293249, which has high homology to known pufferfish urocortin sequences. The full length sequence was amplified from human genomic DNA and sequenced. Sequence homology comparisons of the novel sequence with human urocortin I and urocortin II revealed that the sequence encoded a novel human urocortin, which was designated urocortin III (UcnIII). While urocortin III does not have high affinity for either CRF-R1 or CRF-R2, the affinity for CRF-R2 is greater than the affinity for CRF-R1. Urocortin III is capable stimulating cyclic AMP production in cells expressing CRF-R2α or β. Thus, the affinity is high enough that urocortin III could act as a native agonist of CRF-R2. However, it is also likely that urocortin III is a stronger agonist of a yet to be identified receptor.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This non-provisional application claims benefit of priority ofprovisional U.S. No. 60/276,069, filed Mar. 15, 2001, now abandoned.

FEDERAL FUNDING LEGEND

[0002] This invention was produced in part using funds from the Federalgovernment under grant no P01-DK-26741. Accordingly, the Federalgovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to the fields ofneuroendocrinology and neuropeptide chemistry. More specifically, theinstant invention relates to protein factors involved in the regulationof neuroendocrine and paracrine responses to stress. Most specifically,the present invention discloses a corticotropin releasing factor relatedpeptide designated urocortin III.

[0005] 2. Description of the Related Art

[0006] Corticotropin releasing factor (CRF) and its related family ofpeptides were recognized initially for their regulation of thehypothalamic-pituitary-adrenal axis (HPA) under basal and stressconditions (1, 2). Corticotropin releasing factor (CRF) is a 41 aminoacid peptide that was first isolated from ovine hypothalamus (3) andshown to play an important role in the regulation of thepituitary-adrenal axis, and in endocrine, autonomic and behavioralresponses to stress (4). The CRF family of neuropeptides also includesstructurally related mammalian and non-mammalian peptides such asurocortin (Ucn), a 40 amino acid peptide originally identified in ratbrain (5), fish urotensin I (Uro) (6), and amphibian sauvagine (Svg)(7).

[0007] It has been hypothesized that members of the CRF family areinvolved in neuroendocrine and paracrine responses in many tissues. Inaddition to their effects on the pituitary and central nervous system,members of the CRF family have been shown to modulate cardiovascular andgastrointestinal functions and inflammatory processes in mammals tointegrate endocrine, autonomic and behavioral responses to stressors.These peptides may also be implicated in the control of appetite,arousal, and cognitive functions. Severe psychological and physiologicalconsequences can occur as a result of the long term effects of stress,such as anxiety disorders, anorexia nervosa, gastrointestinaldysfunction and melancholic depression.

[0008] CRF family members mediate their biological actions byspecifically binding to CRF receptors with high affinities (8, 9). CRFreceptors are G-protein coupled receptors that act through adenylatecyclase and are structurally related to the secretin receptor family.This family also includes GRF, VIP, PTH, and the calcitonin receptors.

[0009] The CRF receptors are derived from two distinct genes, CRFreceptor type 1 (CRF-R1) (10-12) and CRF receptor type 2 (CRF-R2)(13-15). CRF-R1 and CRF-R2 have distinct pharmacologies and differ intheir anatomical distribution (16). The type 1 CRF receptor (CRF-R1)gene has 13 exons; several splice variants of this receptor have beenfound. The CRF-R1 is distributed throughout the brain and is found insensory and motor relay sites (17). The rodent type 2α receptor(CRF-R2α) is distributed in lateral septum, ventral medial hypothalamus,nucleus of the solitary tract and the dorsal raphe nucleus, which areareas where CRF-R1 is expressed very little or not at all (18). Therodent type 2β receptor (CRF-R2β) is found mostly in peripheral sitesincluding the heart, blood vessels, gastrointestinal tract, epididymis,lung and skin (9, 19).

[0010] The pharmacology of the two types of receptors differs in thatCRF has a modest affinity for CRF-R2 [Ki=5-100 nM] but high affinity forCRF-R1 [Ki=1-2 nM]. Other related peptides such as carp urotensin, frogsauvagine, and urocortin have a high affinity for both CRF-R1 andCRF-R2. CRF-R2 knockout mice demonstrate an increased anxiety-likebehavior caused by hypersensitivity to stressors (5, 20).

[0011] Recently, searches of the public human genome database identifieda region with significant sequence homology to the CRF neuropeptidefamily. The entire human sequence was amplified and sequenced. The humansequence, however, lacks a consensus proteolytic cleavage site thatwould allow for C-terminal processing of the peptide, and is thereforereferred to as an urocortin-related peptide (URP) sequence. Usinghomologous primers deduced from the human sequence, a mouse cDNA wasisolated from whole brain poly (A+) RNA that encodes a predicted 38amino acid peptide, designated urocortin II, which is structurallyrelated to the other known mammalian family members, CRF and urocortin(Ucn). The question of whether human urocortin-related peptiderepresents the mouse Ucn II ortholog remains open until additional mousegenes are identified. Ucn II binds selectively to the type 2 CRFreceptor (CRF-R2), with no appreciable activity on CRF-R1. Transcriptsencoding Ucn II are expressed in discrete regions of the rodent CNS,including stress-related cell groups in the hypothalamus(paraventricular and arcuate nuclei) and brainstem (locus coeruleus).These findings identify Ucn II as a new member of the CRF family ofneuropeptides, which is expressed centrally and binds selectively toCRF-R2. Initial functional studies are consistent with Ucn IIinvolvement in central autonomic and appetitive control, but not ingeneralized behavioral activation (21).

[0012] The prior art is deficient in the recognition of the humanUrocortin-III gene and protein and uses thereof. The present inventionfulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

[0013] A human urocortin, Urocortin-III (Ucn-III) with homology to knownpufferfish urocortins was identified from the public human genomedatabase. From the sequence of the human gene, a mouse ortholog wasisolated. The present invention relates to these novel genes and usesthereof.

[0014] In one aspect, the instant invention is directed to an isolatedand purified urocortin III protein, which may be either mouse or humanurocortin III. The mouse protein preferably has an amino acid sequenceof SEQ ID No. 5, which is derived from a precursor peptide of SEQ ID No.4. The human protein preferably has an amino acid sequence of SEQ ID No.3 derived from a precursor peptide of SEQ ID No. 2.

[0015] The instant invention is also directed to human urocortin IIIcontaining one or more amino acid substitutions derived from the mouseamino acid sequence. The sequence of mouse urocortin III (SEQ ID No. 5)differs from human urocortin III (SEQ ID No. 3) by four amino acids,specifically Ile₁₄, Asp₁₉, Lys₂₇, and Gln₃₃. Substitution of the Leu₁₄residue in the human protein with Ile is contemplated to be especiallyuseful.

[0016] The instant invention is also directed to a pharmaceuticalcomposition comprising a urocortin III protein and to a method oftreating a pathophysiological state using this pharmaceuticalcomposition. This pharmaceutical composition could be administered toactivate the CRF-R2 receptor to remedy a pathophysiological state suchas high body temperature, appetite dysfunction, congestive heartfailure, vascular disease, stress and anxiety.

[0017] The instant invention is also directed to modification of aurocortin III protein. The N-terminus of urocortin III may be extendedwith additional amino acids or peptides such as Threonine-Lysine (thepreceding two residues in the precursor protein), D-tyrosine,L-tyrosine, D-tyrosine-glycine, or L-tyrosine-glycine. In addition, oneor more methionine residues in urocortin III, such as those at position12 and 35 of SEQ ID No. 3, may be replaced with Nle residues.Alternatively, the N-terminus may be extended with D-iodotyrosine,L-iodotyrosine, D-iodotyrosine-glycine, and L-iodotyrosine-glycine andthe methionine residues at positions 12 and 35 replaced with Nle. Theiodotyrosine residues may be labeled with ¹²⁵I.

[0018] Additional substitutions are suggested by amino acid residuesconserved in other urocortin and urocortin-related proteins which differin urocortin III. Such urocortin analogs may be comprised of urocortinIII with one or more amino acid substitutions selected from the groupconsisting of Ile₃, Nle₃, C_(α)Me-Leu₃, Ile₅, Nle₅, C_(α)Me-Leu₅, Leu₇,Nle₇, Thr₈, Ile₉, Phe₉, Gly₁₀, His₁₀, Leu₁₁, Nle₁₁, Leu₁₂, Nle₁₂, Arg₁₃,Gln13, Nle₁₄, C_(α)Me-Leu₁₄, Nle₁₅, C_(α)Me-Leu₁₅, Leu₁₆, Nle₁₆, Glu₁₇,Asp₁₇, Arg₂₀, Nle₂₄, C_(α)Me-Leu₂₄, Arg₃₂, Ile₃₄, Nle₃₄, C_(α)Me-Leu₃₄,Leu₃₅, Nle₃₅, Asp₃₆, Glu₃₆, and Val₃₈.

[0019] The instant invention is also directed to a CRF-R2 receptorantagonist comprising urocortin III protein or a urocortin III analogwherein the first five to eight N-terminal amino acids of the proteinhave been deleted. This antagonist may be incorporated into apharmaceutical composition and used to treat congestive heart failure,vascular disease, gastrointestinal dysfunction and migraine headaches oras an angiogenesis inhibitor.

[0020] In yet another embodiment of the instant invention, Urocortin IIImay also be modified to contain a fluorescent label or a complexingagent for radionuclides. The resulting labeled urocortin III can be usedto identify cells expressing urocortin III receptors. Alternatively,urocortin III may be linked to a toxin molecule.

[0021] In yet another embodiment of the instant invention, an antibodydirected against urocortin III is provided. In a preferred embodiment,the antibody is a monoclonal antibody. The antibody may be conjugated toa molecular label such as a fluorescent label, photoaffinity label orradioactive markers. Alternatively, the antibody could be conjugated toa cytotoxic compound to form an immunotoxin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] So that the matter in which the above-recited features,advantages and objects of the invention, as well as others which willbecome clear, are attained and can be understood in detail, moreparticular descriptions of the invention briefly summarized above may behad by reference to certain embodiments thereof which are illustrated inthe appended drawings. These drawings form a part of the specification.It is to be noted, however, that the appended drawings illustratepreferred embodiments of the invention and therefore are not to beconsidered limiting in their scope.

[0023]FIG. 1 shows the nucleotide and peptide sequences of humanurocortin III.

[0024]FIG. 2A shows the predicted amino-acid sequence encoding human UcnIII while FIG. 2B shows the amino acid sequence of mouse Ucn III. Aminoacids are numbered starting with the initiating methionine. The putativemature peptide coding region is indicated in the boxed area. Thecomplete nucleotide sequences have been deposited with Genbank(accession nos. AF361943 for human Ucn III and AF361944 for mouse UcnIII).

[0025]FIG. 2C shows the alignment of putative mature peptide regions ofhuman and mouse Ucn III with homologous pufferfish urocortins, human andmouse Ucn II, human and ovine CRF, pufferfish urotensin (Uro), frogsauvagine, human and mouse Ucn. Residues identical to human Ucn IIIsequence are boxed. Alignment was made using the Clustal Method ofMegalign in DNASTAR. ▪, Amidation site (putative for human Ucn II).

[0026]FIG. 2D shows a phylogenetic tree which groups human and mouse UcnIII with the pufferfish urocortins and human and mouse Ucn II. The moredistantly related group is comprised of ovine and human CRF; human andmouse Ucn, pufferfish Uro and frog sauvagine. The scale beneath the treemeasures sequence distances. The phylogenetic tree was generated byDNASTAR.

[0027]FIGS. 3A and 3B shows the effects of Ucn related peptides on cAMPaccumulation in a CRF R2β expressing cell line (FIG. 3A) and primary ratanterior pituitary cells (FIG. 3B). FIG. 3A shows results from A7r5 rataortic smooth muscle cells. EC₅₀: mUcn II: 0.18 nM; mUcn III: 3.7 nM;hUcn III: 80.9 nM. FIG. 3B shows results from primary rat anteriorpituitary cells which were established in culture and were stimulatedwith various peptides for 45 min. EC₅₀: rUcn: 2.3 nM; hUcn II: 1 μM*,mUcn II: 0.75 μM* (*: estimated using the plateau of rUcn).

[0028]FIG. 4 show the expression of mouse Ucn III mRNA in brain andperipheral tissues. A representative image of RNase protection assay ofUcn III mRNA is shown. Total RNA isolated from each tissue listed washybridized with the mouse Ucn III antisense probe and mouse GAPDH. Theprotected fragments were resolved on a 6% polyacrylamide urea gel.Abbreviations: BnST: bed nucleus of stria terminalis.

[0029] FIGS. 5A-5F show hybridization histochemical localization of UcnIII mRNA in the rat brain. Positive hybridizing signal was mostprominent in three regions of the ventral forebrain. These included themedian preoptic nucleus (FIGS. 5A, 5B), the rostral perifornical areawhich encompasses areas just lateral to the paraventricular nucleus(FIG. 5C), and the posterior part of the bed nucleus of stria terminalis(FIG. 5D), and the medial amygdaloid nucleus (FIG. 5E). In the brainstem, positive hybridization signals were detected mainly in thesuperior paraolivary nucleus (FIG. 5F). Abbreviations: 3V: thirdventricle; ac: anterior commissure; BSTp: posterior part of the bednucleus of stria terminalis; fx: fornix; MeA: medial nucleus ofamygdala; MePO: median preoptic nucleus; OVLT: vascular organ of thelamina terminalis; opt: optic tract; PVH: paraventricular nucleus ofhypothalamus; SPO: superior paraolivary nucleus; Tz: nucleus of thetrapezoid body. Scale bars=50 μm.

DETAILED DESCRIPTION OF THE INVENTION

[0030] In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques, all within the skill of the art. Such techniques areexplained fully in the literature. See, e.g., Maniatis, Fritsch &Sambrook, “Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning:A Practical Approach,” Volumes I and II (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins Eds. (1985)]; “Transcriptionand Translation” [B. D. Hames & S. J. Higgins Eds. (1984)]; “Animal CellCulture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes”[IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning”(1984). Other employed techniques may be peptide synthetic (Stewart, J.M.; Young, J. D. Solid Phase Peptide Synthesis. In Solid Phase PeptideSynthesis; Eds.; Pierce Chemical Co.: Rockford, Ill., 1984; V. pp 176),analytical chemistry (Miller, C.; Rivier, J. Peptide chemistry:Development of high-performance liquid chromatography and capillary zoneelectrophoresis. Biopolymers 1996, 40, 265-317.), structure activityrelationship approaches (including in vivo and in vitro testing andstructural analysis using NMR, CD, X-ray crystallography among others)(Gulyas, J.; Rivier, C.; Perrin, M.; Koerber, S. C.; Sutton, S.;Corrigan, A.; Lahrichi, S. L.; Craig, A. G.; Vale, W. W.; Rivier, J.Potent, structurally constrained agonists and competitive antagonists ofcorticotropin releasing factor (CRF). Proc. Natl. Acad. Sci. USA 1995,92, 10575-10579).

[0031] Therefore, if appearing herein, the following terms shall havethe definitions set out below.

[0032] As used herein, the term “cDNA” shall refer to the DNA copy ofthe mRNA transcript of a gene.

[0033] As used herein, the term “derived amino acid sequence” shall meanthe amino acid sequence determined by reading the triplet sequence ofnucleotide bases in the cDNA.

[0034] As used herein the term “screening a library” shall refer to theprocess of using a labeled probe to check whether, under the appropriateconditions, there is a sequence complementary to the probe present in aparticular DNA library. In addition, “screening a library” could beperformed by PCR.

[0035] As used herein, the term “PCR” refers to the polymerase chainreaction that is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202to Mullis, as well as other improvements now known in the art.

[0036] It should be noted that all amino-acid residue sequences arerepresented herein by formulae whose left and right orientation is inthe conventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues.

[0037] The amino acids described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property of immunoglobulin binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J Biol. Chem., 243:3552-59 (1969),abbreviations for amino acid residues are known in the art.

[0038] Nonstandard amino acids may be incorporated into proteins bychemical modification of existing amino acids or by de novo synthesis ofa protein/peptide. A Nonstandard amino acid refers to an amino acid thatdiffers in chemical structure from the twenty standard amino acidsencoded by the genetic code. Post-translational modification in vivo canalso lead to the presence of a nonstandard or amino acid derivative in aprotein. The N-terminal NH₂ and C-terminal COOH groups of a protein canalso be modified, for example, by natural or artificialpost-translational modification of a protein.

[0039] Proteins/peptides may be modified by amino acids substitutions.Often, some changes result in significant changes in the activity(agonists versus antagonists) and potency/affinity of proteins/peptideswhile other have little or no effect. Conservative substitutions areleast likely to drastically alter the activity of a protein. A“conservative amino acid substitution” refers to replacement of aminoacid with a chemically similar amino acid, i.e. replacing nonpolar aminoacids with other nonpolar amino acids; substitution of polar amino acidswith other polar amino acids, acidic residues with other acidic aminoacids, etc. Examples of preferred conservative substitutions are setforth in Table I: TABLE 1 Conservative Amino Acid Substitutions MostPreferred Original Preferred Conservative Conservative ResidueSubstitutions Substitution Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln;Asn Lys Asn (N) Gln; His; Lys; Arg, Ser Gln Asp (D) Glu Glu Cys (C) SerSer Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala, DAla Pro His (H)Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Nle Leu Leu (L)Ile; Val; Met; Ala; Phe; Nle Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu;Phe; Ile, Nle Leu Phe (F) Leu; Val; Ile; Ala Leu Pro (P) Gly, Sar GlySer (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Nal, Cpa Tyr Tyr (Y) Trp;Phe; Thr; Ser, His Phe Val (V) Ile; Leu; Met; Phe; Ala; Nle Leu

[0040] “Chemical derivative” refers to a subject polypeptide having oneor more residues chemically derivatized by reaction of a functional sidegroup. Such derivatized polypeptides include, for example, those inwhich free amino groups have been derivatized to form specific salts orderivatized by alkylation and/or acylation, p-toluene sulfonyl groups,carbobenzoxy groups, t-butylocycarbonyl groups, chloroacetyl groups,formyl or acetyl groups among others. Free carboxyl groups may bederivatized to form organic or inorganic salts, methyl and ethyl estersor other types of esters or hydrazides and preferably amides (primary orsecondary). Chemical derivatives may include those peptides whichcontain one or more naturally occurring amino acids derivatives of thetwenty standard amino acids. For example, 4-hydroxyproline may besubstituted for serine; and ornithine may be substituted for lysine.Peptides embraced by the present invention also include peptides havingone or more residue additions and/or deletions relative to the specificpeptide whose sequence is shown herein, so long as the modified peptidemaintains the requisite biological activity.

[0041] A “replicon” is any genetic element (e.g., plasmid, chromosome,virus) that functions as an autonomous unit of DNA replication in vivo;i.e., capable of replication under its own control.

[0042] A “vector” is a replicon, such as plasmid, phage or cosmid, towhich another DNA segment may be attached so as to bring about thereplication of the attached segment.

[0043] A “DNA molecule” refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itseither single stranded form, or a double-stranded helix. This termrefers only to the primary and secondary structure of the molecule, anddoes not limit it to any particular tertiary forms. Thus, this termincludes double-stranded DNA found, inter alia, in linear DNA molecules(e.g., restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA).

[0044] An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

[0045] A DNA “coding sequence” is a double-stranded DNA sequence, whichis transcribed and translated into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

[0046] Transcriptional and translational control sequences are DNAregulatory sequences, such as promoters, enhancers, polyadenylationsignals, terminators, and the like, that provide for the expression of acoding sequence in a host cell.

[0047] A “promoter sequence” is a DNA regulatory region capable ofbinding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded at its 3′ terminusby the transcription initiation site and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site, as well asprotein binding domains (consensus sequences) responsible for thebinding of RNA polymerase. Eukaryotic promoters often, but not always,contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters containShine-Dalgarno sequences in addition to the −10 and −35 consensussequences.

[0048] An “expression control sequence” is a DNA sequence that controlsand regulates the transcription and translation of another DNA sequence.A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

[0049] A “signal sequence” can be included near the coding sequence.This sequence encodes a signal peptide, N-terminal to the polypeptide,which communicates to the host cell to direct the polypeptide to thecell surface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

[0050] The term “oligonucleotide”, as used herein in referring to theprobe of the present invention, is defined as a molecule comprised oftwo or more ribonucleotides, preferably more than three. Its exact sizewill depend upon many factors which, in turn, depend upon the ultimatefunction and use of the oligonucleotide.

[0051] The term “primer” as used herein refers to an oligonucleotide,whether occurring naturally as in a purified restriction digest orproduced synthetically, which is capable of acting as a point ofinitiation of synthesis when placed under conditions in which synthesisof a primer extension product, which is complementary to a nucleic acidstrand, is induced, i.e., in the presence of nucleotides and an inducingagent such as a DNA polymerase and at a suitable temperature and pH. Theprimer may be either single-stranded or double-stranded and must besufficiently long to prime the synthesis of the desired extensionproduct in the presence of the inducing agent. The exact length of theprimer will depend upon many factors, including temperature, source ofprimer and use the method. For example, for diagnostic applications,depending on the complexity of the target sequence, the oligonucleotideprimer typically contains 15-25 or more nucleotides, although it maycontain fewer nucleotides.

[0052] The primers herein are selected to be “substantially”complementary to different strands of a particular target DNA sequence.This means that the primers must be sufficiently complementary tohybridize with their respective strands. Therefore, the primer sequenceneed not reflect the exact sequence of the template. For example, anon-complementary nucleotide fragment may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence beingcomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementary with the sequence orhybridize therewith and thereby form the template for the synthesis ofthe extension product.

[0053] As used herein, the terms “restriction endonucleases” and“restriction enzymes” refer to enzymes, each of which cutdouble-stranded DNA at or near a specific nucleotide sequence.

[0054] A cell has been “transformed” by exogenous or heterologous DNAwhen such DNA has been introduced inside the cell. The transforming DNAmay or may not be integrated (covalently linked) into the genome of thecell. In prokaryotes, yeast, and mammalian cells for example, thetransforming DNA may be maintained on an episomal element such as aplasmid. With respect to eukaryotic cells, a stably transformed cell isone in which the transforming DNA has become integrated into achromosome so that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the transforming DNA. A “clone”is a population of cells derived from a single cell or ancestor bymitosis. A “cell line” is a clone of a primary cell that is capable ofstable growth in vitro for many generations.

[0055] Two DNA sequences are “substantially homologous” when at leastabout 75% (preferably at least about 80%, and most preferably at leastabout 90% or 95%) of the nucleotides match over the defined length ofthe DNA sequences. Sequences that are substantially homologous can beidentified by comparing the sequences using standard software availablein sequence data banks, or in a Southern hybridization experiment under,for example, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

[0056] A “heterologous’ region of the DNA construct is an identifiablesegment of DNA within a larger DNA molecule that is not found inassociation with the larger molecule in nature. Thus, when theheterologous region encodes a mammalian gene, the gene will usually beflanked by DNA that does not flank the mammalian genomic DNA in thegenome of the source organism. In another example, the coding sequenceis a construct where the coding sequence itself is not found in nature(e.g., a cDNA where the genomic coding sequence contains introns orsynthetic sequences having codons different than the native gene).Allelic variations or naturally occurring mutational events do not giverise to a heterologous region of DNA as defined herein.

[0057] The labels most commonly employed for these studies areradioactive elements, enzymes, chemicals that fluoresce when exposed toultraviolet light, and others. A number of fluorescent materials areknown and can be utilized as labels. These include, for example,fluorescein, rhodamine, auramine, Texas Red, AMCA blue and LuciferYellow. A particular detecting material is anti-rabbit antibody preparedin goats and conjugated with fluorescein through an isothiocyanate.

[0058] A particular assay system developed and utilized in the art isknown as a receptor assay. In a receptor assay, the material to beassayed is appropriately labeled and then certain cellular test coloniesare inoculated with a quantity of both the labeled and non-labeledmaterial after which binding studies are conducted to determine theextent to which the labeled material binds to the cell receptors. Inthis way, differences in affinity between materials can be ascertained.

[0059] As used herein, the term “host” is meant to include not onlyprokaryotes but also eukaryotes such as yeast, plant and animal cells. Arecombinant DNA molecule or gene that encodes a protein of the presentinvention can be used to transform a host using any of the techniquescommonly known to those of ordinary skill in the art. Prokaryotic hostsmay include E. coli, S. tymphimurium, Serratia marcescens and Bacillussubtilis. Eukaryotic hosts include yeasts such as Pichia pastoris,mammalian cells and insect cells.

[0060] In general, expression vectors containing promoter sequences thatfacilitate the efficient transcription of the inserted DNA fragment areused in connection with the host. The expression vector typicallycontains an origin of replication, promoter(s), terminator(s), as wellas specific genes that are capable of providing phenotypic selection intransformed cells. The transformed hosts can be fermented and culturedaccording to means known in the art to achieve optimal cell growth.

[0061] Methods well known to those skilled in the art can be used toconstruct expression vectors containing appropriate transcriptional andtranslational control signals. See for example, the techniques describedin Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (2ndEd.), Cold Spring Harbor Press, N.Y. A gene and its transcriptioncontrol sequences are defined as being “operably linked” if thetranscription control sequences effectively control the transcription ofthe gene. Vectors of the invention include, but are not limited to,plasmid vectors and viral vectors.

[0062] In one embodiment of the instant invention, an isolated andpurified urocortin III protein is provided. This protein may be eitherthe human or mouse urocortin III protein. The human protein is encodedby DNA partially comprised by a human EST, GenBank accession numberAW293249 with significant sequence homology to the pufferfishurocortins.

[0063] Another embodiment of the instant invention is directed to ahuman urocortin III protein encoded by a precursor peptide of SEQ ID No.2. After post-translational modification, the purified human urocortinIII preferably has an amino acid sequence corresponding to SEQ ID No: 3.

[0064] The instant invention is also directed to modification of theurocortin III proteins. The N-terminal ends of the urocortin III protensmay be modified with various acylating agents such ascarboxyl-containing moieties, sulfonyl-containing moieties andisocyanates. Alternatively, the N-terminal end of urocortin III may bechemically crosslinked to a toxin molecule. The N-terminus of urocortinIII may also be extended with additional amino acids or peptides such asD-tyrosine, L-tyrosine, D-tyrosine-glycine, or L-tyrosine-glycine. Inaddition, one or more methionine residues in urocortin III, such asthose at position 12 and 35 of SEQ ID No. 3, may be replaced with Nleresidues. Alternatively, the N-terminus may be extended withD-iodotyrosine, L-iodotyrosine, D-iodotyrosine-glycine, andL-iodotyrosine-glycine and the methionine residues at positions 12 and35 replaced with Nle. The iodotyrosine residues may be labeled with¹²⁵I.

[0065] In another embodiment of the instant invention, pharmaceuticalcomposition comprising a urocortin III protein is provided as well as amethod of treating a pathophysiological state using this pharmaceuticalcomposition. The pharmaceutical composition may be administered, forexample, to activate the CRF-R2 receptor in an individual and can thusremedy various pathophysiological states such as high body temperature,appetite dysfunction, congestive heart failure, vascular disease, stressand anxiety.

[0066] Another embodiment of the instant invention is directed tomodification of a urocortin III protein. The N-terminus of urocortin IIImay be extended with additional amino acids or peptides such asD-tyrosine, L-tyrosine, D-tyrosine-glycine, or L-tyrosine-glycine. Inaddition, one or more methionine residues in urocortin III, such asthose at position 12 and 35 of SEQ ID No. 3, may be replaced with Ile,Val, Leu or preferably Nle residues. Alternatively, the N-terminus maybe extended with D-iodotyrosine, L-iodotyrosine, D-iodotyrosine-glycine,and L-iodotyrosine-glycine and the methionine residues at positions 12and 35 replaced with Nle. The iodotyrosine residues may be labeled with¹²⁵I.

[0067] Additional substitutions are suggested by amino acid residuesconserved in other urocortin and urocortin-related proteins which differin urocortin III. Such urocortin analogs may be comprised of urocortinIII with one or more amino acid substitutions selected from the groupconsisting of Ile₃, Nle₃, C_(α)Me-Leu₃, Ile₅, Nle₅, C_(α)Me-Leu₅, Leu₇,Nle₇, Thr₈, Ile₉, Phe₉, Gly₁₀, His₁₀, Leu₁₁, Nle₁₁, Leu₁₂, Nle₁₂, Arg₁₃,Gln₁₃, Nle₁₄, C_(α)Me-Leu₁₄, Nle₁₅, C_(α)Me-Leu₁₅, C_(α)Me-Leu₁₆, Leu₁₆,Nle₁₆, Glu₁₇, Asp₁₇, Nle₁₈, Leu₁₈, Arg₂₀, Nle₂₄, C_(α)Me-Leu₂₄, Arg₃₂,Ile₃₄, Nle₃₄, C_(α)Me-Leu₃₄, Leu₃₅, Nle₃₅, AsP₃₆, Glu₃₆ and Val₃₈.

[0068] In yet another embodiment of the instant invention, a CRF-R2receptor antagonist is provided. This antagonist comprises urocortin IIIprotein or urocortin III analog wherein the first five to eightN-terminal amino acids of the protein have been deleted. This antagonistmay be incorporated into a pharmaceutical composition and used to treatcongestive heart failure, vascular disease, gastrointestinal dysfunctionand migraine headaches or may be used as an angiogenesis inhibitor.

[0069] In a further embodiment of the instant invention, Urocortin IIIis modified to contain a fluorescent label or a complexing agent forradionuclides. The resulting labeled urocortin III can be used toidentify cells expressing urocortin III receptors. Alternatively,urocortin III may be linked to a toxin molecule.

[0070] In yet another embodiment of the instant invention, an antibodydirected against urocortin III is provided. In a preferred embodiment,the antibody is a monoclonal antibody. The antibody may be conjugated toa molecular label such as a fluorescent label, photoaffinity label orradioactive markers.

[0071] The following examples are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion.

EXAMPLE 1 Identification of Human Urocortin III

[0072] The human expressed sequence tag (EST) database of Genbank wassearched using a pufferfish (Takifugu rubripes) sequence (Genbankassession number AJ251323) related to urocortin (urocortin relatedpeptide, URP) as a probe. This search identified a human EST, GenBankaccession number AW293249 with significant sequence homology to thepufferfish urocortins. Sequence homology comparisons of the novelsequence with human urocortin I and urocortin II revealed that thesequence encoded a novel human urocortin, which was designated urocortinIII. The partial human EST contained the precursor sequence and thefirst 29 amino acids of the mature peptide region. Nested primers forurocortin III were designed based on the partial human EST sequenceconsisting of the sequences 5′-AAG AGT CCC CAC CGC ACC AAG TTC ACC-3′(SEQ ID No. 16) and 5′-TCC CTC GAC GTC COC ACC AAC ATC ATG-3′ (SEQ IDNo. 17). These primers were used along with nested anchored primers toscreen a Human GenomeWalker Kit (Clontech) by PCR. Nested PCR wasperformed for 35 cycles consisting of denaturation at 95° C. for fiveminutes followed by sequence extension at 66° C. for 12 min. Theamplified fragments were subcloned into pCRIITOPO vector (Invitrogen),sequenced and found to encode a full-length mature peptide.

[0073] To extend sequence information at the C-terminus, primers weredesigned based on the partial human EST sequence to screen a humanGenomeWalker Kit (Clontech) by PCR. Library pools of human genomic DNAwere screened using gene specific and anchored primers in a nested PCRstrategy. A full-length human gene encoding a protein for a putativepeptide was identified. This protein, named urocortin III (Ucn III), isrelated to other known CRF family members.

EXAMPLE 2 Identification of Mouse Urocortin III by HybridizationScreening

[0074] Based on the full-length human Ucn III sequence, a 144 bp probewas generated, spanning the mature peptide region, to search for a mouseortholog. The probe was used to screen a mouse genomic λFIXII library(Stratagene) by low stringency hybridization. Hybridization was carriedout at 42° C. overnight, in 20% formamide/5×SSC/5×Denhardts/0.5% SDS/5%dextran sulfate. Washes were performed in 1×SSC at 55° C. Purifiedplaques were subcloned into pBluescript and sequenced. A full-lengthmouse genomic clone was identified and sequenced. A mouse Ucn III cDNAwas isolated from whole brain cDNA by PCR using primers designed fromthe mouse genomic sequence. PCR was performed at 55° C. for 35 cycleswith 2 min extension at 72° C.

EXAMPLE 3 Analysis of the Human and Mouse Ucn III Genes

[0075] Both the human and mouse Ucn III genes contain two potentialinitiation sites for translation. The nucleotide sequence of the humangene encodes a protein deduced to be either 161 or 159 amino acids,depending on the N-terminal methionine used, with preference for thefirst methionine according to the Netstart 1.0 prediction server(22)(FIG. 2A). The mouse gene encodes a protein deduced to be either a164 or 162 amino acid precursor also with preference for translationbeginning at the first methionine (FIG. 2B). Processing of the precursormolecule to generate the putative 38 amino acid mature peptide ispredicted to occur by cleavage at the C-terminal side of Lys 119 for thehuman gene and following Lys 122 for mouse Ucn III. Thus, Ucn IIIconforms to the rules for processing at monobasic residues (23), similarto Ucn and murine Ucn II. The C-terminal sequence contains a pair ofbasic residues, (R-K) for the human and (K-K) for the mouse, immediatelypreceded by a glycine, presumed to be involved in C-terminal amidation.The predicted mature peptide regions of the human and mouse Ucn IIIpeptides are shown in the boxed regions (FIGS. 2A and 2B). Because themature peptide regions of human and mouse Ucn III differ by only 4 aminoacid residues, they are likely to be orthologous. These sequences havebeen deposited in the Genbank database [accession nos AF361943 (humanUcn III) and AF361944 (mouse Ucn III)].

EXAMPLE 4 Comparison of Ucn III to other CRF Family Members

[0076] In FIG. 2C, the 38 amino acid mature peptide region of human UcnIII sequence is compared to that of other family members. Human andmouse Ucn III share 40% identity to mouse Ucn II. Human and mouse UcnIII share 37% identity to human URP. They are more distantly related toUcn and CRF. Human and mouse Ucn III share 21% and 18% identity withhuman and mouse Ucn, respectively. Human and mouse Ucn III share 32% and26% identity to human/rat CRF. The mammalian urocortins II and IIIappear to be a separate but related evolutionary branch of theCRF-family with closer ties to two pufferfish urocortins than tomammalian Ucn or CRF (FIG. 2D). Three CRF-family related pufferfishsequences exist to date in the Genbank database. At the amino acidlevel, the pufferfish urocortin related peptide (URP) (Genbank accessionno. AJ251323) is most closely related (76% identity) to both human andmouse Ucn III, and more distantly related to human URP (42%) and mouseUcn II (37%). A second pufferfish urocortin sequence (Genbank AL175143)is also more closely related to Ucn III (53%) than to any of the othermammalian CRF family members. The third pufferfish peptide (GenbankAL218869) is most similar to the fish urotensin I's with highest aminoacid identity to flounder urotensin I (63%).

EXAMPLE 5 Synthesis of Urocortin III

[0077] Human and mouse Ucn III were synthesized manually using the solidphase approach, with a 4-methylbenzhydrylamine resin and theBoc-strategy (24). Trifluoroacetic acid (TFA 60% in dichloromethane) wasused to remove the Boc groups. Main chain assembly was performed usingdiisopropylcarbodiimide. The peptide was cleaved and deprotected inhydrofluoric acid and purified using RP-HPLC and three solvent systems(triethylammonium phosphate at pH 2.25 and pH 6.5 and/or 0.1%TFA) (25).Peptides were greater than 95% pure using independent HPLC and capillaryzone electrophoresis criteria. Mass spectra confirmed the composition ofthe preparations.

[0078] In addition to the synthesized Urocortin III (calc. Mass 4136.34,found 4136.2) described above, the following synthetic analog were alsoconstructed by the same method: [Tyr-Gly]-Urocortin III (calc Mass4356.34 found 4356.3); [Nle_(12,35)]-Urocortin III (calc. Mass 4100.34found 4100.2); [Tyr₀,Nle_(12,35)]-Urocortin III (calc. Mass 4263.41,found 4263.2) and [Tyr-Gly₀,Nle_(12,35)]-Urocortin III (calc. Mass4320.431, found 4320.4).

EXAMPLE 6 Ucn III Binding Assays

[0079] The synthesized urocortin III was assayed for binding to theCRF-R1, CRF-R2α and CRF-R2β receptors by competitive displacement of²⁵¹I[Tyr⁰,Glu¹,Nle₁₇]sauvagine from Chinese hamster ovary cells stablyexpressing each receptor type. Stably transfected cell lines expressingCRF-R2α were generously provided by Demitri Grigoriadis, NeurocrineBiosciences Inc. Crude membrane fractions were prepared from Chinesehamster ovary cells stably expressing either cloned human CRF-R1 ormouse CRF-R2β as described (5). Test peptides and radio-ligand,[¹²⁵ITyr⁰,Glu¹,Nle¹⁷]-sauvagine, diluted in assay buffer (20 mM Hepes/2mM EGTA/0.1% BSA/10% sucrose, pH 7.6), were combined with membranefractions (10 μg) in MAGV microtiter plates (Millipore) precoated with0.1% polyethyleneimine. After 90 min at room temperature, the reactionmixture was filtered and washed twice with assay buffer. The radioligandcomplex was quantified by gamma-counting. Inhibitory binding constants,K_(i)'s, were determined by GRAPHPAD. The affinities of Ucn II and UcnIII for the CRF binding protein (CRF-BP) were estimated on the basis ofthe displacement of [D¹²⁵ITyr⁰]-hCRF according to the techniquedescribed in reference (26).

[0080] Data from at least 3 experiments were pooled and inhibitorydissociation constant (K_(i)) values were calculated using the GraphpadPrism program EC₅₀ values. The average of their log₁₀ value wascalculated and used to exponentiate the number 10 to determine an‘average’ EC₅₀ value. The error was determined by calculating thestandard deviation of the log₁₀ values, by which the number 10 wasexponentiated to determine an error factor. The calculated EC50 was thendivided or multiplied by the error factor to determine the lower andupper error bounds, respectively.

[0081] Comparisons of the binding affinities and potencies forstimulating cAMP accumulation in cells stably expressing the humanCRF-R1 , rat CRF-R2α and mouse CRF-R2β are shown in Table 2. Ucn issignificantly more potent than either Ucn II or Ucn III in binding toCRF-R1. Both Ucn II and Ucn III selectively bind both splice variants ofCRF-R2, compared to CRF-R1. Human Ucn III displays significantly loweraffinity for either type 2 receptor than Ucn or Ucn II. Mouse Ucn IIIdisplays considerably higher affinity for murine CRF-R2 receptors thandoes human Ucn III. Both Ucn II and Ucn III display slightly higheraffinities for CRF-R2β compared to CRF-R2α. The potency advantage ofmouse Ucn III over human Ucn III is also observed for binding to humanCRF-R2α (data not shown). TABLE 2 Binding Properties and FunctionalActivities of CRF-family ligands Binding Binding Binding to Membranesfrom to Membranes from to Membranes from CHO cells stably CHO cellsstably CHO cells stably cAMP in CHO cells cAMP in CHO cells cAMP in CHOcells expressing expressing expressing stably expressing stablyexpressing stably expressing hCRF-R1 rCRF-R2α mCRF-R2β hCRF-R1 rCRF-R2αmCRF-R2β Peptide (K_(i), nM) (K_(i), nM) (K_(i), nM) (EC₅₀, nM) (EC₅₀,nM) (EC₅₀, nM) CRF 0.53 10.1  5.2  0.035 0.64 0.42 (rat/human)(0.25-1.15)  (6.5-15.6) (1.6-17)  (0.014-0.082)  (0.03-11.34)(0.17-0.98) Urocortin 0.32 2.2 0.62 0.15   0.063  0.087 (rat)(0.14-0.77) (0.91-5.4)  (0.14-2.8)  (0.03-0.64) (0.014-0.28) 0.017-0.43)  Urocortin II >100 1.7 0.50 >100 0.26 0.42 (human)(0.73-4.1)  (0.22-1.16) (0.11-0.61) 0.16-1.1)  Urocortin II >100 2.10.66 >100 0.14 0.05 (mouse) (0.78-5.4)  (0.13-3.3)  (0.04-0.43)(0.02-0.12) Urocortin III >100 21.7  13.5 >100 0.16 0.12 (human)(8.2-57)   (9.2-19.7) (0.09-0.28) (0.06-0.20) Urocortin III >100 5.01.8  >100  0.073  0.081 (mouse) (4.0-6.3) (0.77-4.1)  (0.052-0.10) (0.08-0.80) #10^(γ)/10^(δ)]. Results are expressed as the average ± semfor three or more independent assays.

EXAMPLE 7 Activation of Adenylate Cyclase in Receptor-Transfected Cells

[0082] Chinese hamster ovary cells stably transfected with either humanCRF-R1 or murine CRF-R2 were plated into 48-well tissue culture dishes(Costar) and allowed to recover for 24 h. The medium was changed atleast 2 h before treatments to DMEM/0.1% FBS. The cells werepreincubated with 0.1 mM 3-isobutyl-1-methylxanthine for 30 min and thenexposed to peptides for 20 min at 37° C. Intracellular cAMP accumulationin CHO cells stably transfected with either CRF-R1 or CRF-R2 was used asa measure of receptor activation. Both Ucn II and Ucn III have very lowpotencies to activate CRF-R1 (>100 nM), contrasting sharply with that ofUcn, whose EC50 is ˜0.15 nM. Indeed, Ucn III shows no activation ofCRF-R1 even at very high doses (1 μM). The potencies of Ucn II and IIIto activate CRF-R2α and CRF-R2β are approximately equal and nearlyequivalent to that of Ucn. Thus, in the cAMP stimulation assay, both UcnII and III show 'selectivity for CRF-R2 over CRF-R1, but no preferencewith respect to CRF-R2α and CRFR-2β. Further, the relative potencies ofmurine and human Ucn II and Ucn III to functionally activate CRF-R2overlap in spite of the lower affinity of human Ucn III for binding toCRF-R2.

EXAMPLE 8 Activation of Adenylate Cyclase in Cells Expressing EndogenousReceptors

[0083] The abilities of Ucn II and Ucn III to activate adenylate cyclasein cells expressing endogenous CRF-R1 (cultured primary anteriorpituitary cells, (5)) or CRF-R2β (A7r5 cells, (27)). Rat aortic smoothmuscle cells, A7r5, were plated into 48-well culture dishes and allowedto recover for 48 h. Cells were starved in DMEM/0.2% FBS overnightbefore the experiment. The cells were preincubated with 0.1 mM3-isobutyl-1-methylxanthine for 30 min and then exposed to peptides for20 min at 37° C. Rat anterior pituitary cells were established inculture (28) and treated with test peptides for 45 min at 37° C.Intracellular cAMP was extracted from all cells and measured fromtriplicate wells using a radioimmunoassay kit (Biomedical Technologies).Potencies were determined using the PRISM GRAPHPAD. The results areshown in FIG. 3.

[0084] In keeping with the results on transfected receptors, Ucn II andUcn III are able to activate endogenous CRF-R2β at sub- or low nanomolarconcentrations of ligand (FIG. 3A). Expectedly, Ucn II exhibits lowpotency to increase cyclic AMP in cultured pituitary cells expressingCRF-R1, whereas Ucn III is inactive in this assay even atconcentrations >1 μM (FIG. 3B).

EXAMPLE 9 Binding to CRF Binding Protein

[0085] As opposed to CRF and urocortin which have subnanomolaraffinities for CRF-BP, neither Ucn II nor Ucn III exhibit appreciableaffinity for this protein (data not shown). Urocortin III does not havea high affinity for either CRF-R1 or CRF-R2. However, the affinity forCRF-R2 is greater than the affinity for CRF-R1. In view of its highpotency to stimulate cyclic AMP production in cells expressing CRF-R2αor β, the affinity is obviously high enough that urocortin III could actas a native agonist of CRF-R2. It is also likely that urocortin III is astronger agonist of a yet to be identified receptor.

EXAMPLE 10 Ucn III mRNA Expression

[0086] RNase protection assays were performed to determine the tissuedistribution of mouse Ucn III mRNA. RNase protection analysis wascarried out as previously described (29). Total RNA was extracted usingTRI REAGENT (Molecular Research Center, Inc., Cincinnati, Ohio). MouseUcn III and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levelswere measured simultaneously by RNase protection, using mouse GAPDH asan internal loading control.

[0087] A 528-nucleotide Ucn III antisense riboprobe specific to themouse Ucn III mRNA was synthesized using T3 RNA polymerase. A200-nucleotide antisense riboprobe specific to mouse GAPDH mRNA wassynthesized using T3 RNA polymerase. All riboprobes were synthesized inthe presence of [α-³²P]UTP (3,000 Ci/mmol) and either 20 μM UTP for UcnIII or 200 μM UTP for GAPDH, as described (29). The fragments sizesprotected by Ucn III and GAPDH riboprobes are 415 and 135 nucleotides,respectively.

[0088] RNA samples (50 μg of peripheral tissue or 20-25 μg of braintissues) were hybridized in 24 μl deionized formamide plus 6 μlhybridization buffer containing 10⁷ cpm of Ucn III and 3×10⁴ cpm GAPDHantisense riboprobes. After heating to 85° C. for 5 min, the sampleswere hybridized at 42° C. for 12 h and subsequently digested by RNase(175 μg/ml RNase A and 500 U/ml RNase T1) at 24° C. for 60 min. Thesamples were resolved on 5% polyacrylamide urea gels. Image analysis wasperformed using the PhosphorImager system (Molecular Dynamics, Inc.,Sunnyvale, Calif.) and the ImageQuant 4.0 software package.

[0089] Total RNA from tissue encompassing several regions of the CNS andperipheral tissues was hybridized with the 528 bp cRNA. This probespanned the mature peptide region and gave a protected fragment of 415bp. In the CNS, sites of mRNA expression include the hypothalamus,brainstem, and lateral septum (LS)/bed nucleus of stria terminalis(BnST) (FIG. 4). The pituitary, cerebellum and cerebral cortex showed nodetectable mRNA expression. In the periphery, Ucn III mRNA is expressedin small intestine and skin, with no detectable expression in heart,aortic vessel, liver or lung (FIG. 4).

EXAMPLE 11 In situ Hybridization

[0090] To reveal a more detailed pattern of expression of Ucn III in thebrain, in situ hybridization was performed on a series of both rat andmouse brain sections using antisense and sense cRNA probes generatedfrom a 415 bp mouse Ucn III cDNA template. Brains and peripheral tissuesobtained from adult mice (C57BL/6) and Sprague-Dawley rats were quicklyremoved and frozen on dry ice. Frozen sections were cut to 20 μm-thickon a cryostat, thaw-mounted onto glass slides, and stored at −80° C.until use. In situ hybridization was performed with ³³P-labeledantisense and sense (control) cRNA probes transcribed from linearizedplasmid (pCRIITOPO) containing the mouse Ucn III cDNA (30). Probes werelabeled to specific activities of 1-3×10⁹ dpm/μg, applied to slides at aconcentration of about 2.8×10⁷ cpm/ml and hybridized overnight at 56° C.Slides were washed in SSC of increasing stringency, followed by RNasetreatment at 37° C., and finally 0.1×SSC at 65° C. Followingdehydration, the slides were exposed to X-ray film (β-Max; Kodak) for 4days at 4° C. and then coated with Kodak NTB-2 liquid emulsion andexposed at 4° C. for 10 days.

[0091] Positive Ucn III mRNA signal was observed only in sectionshybridized with the antisense probe. The distribution of Ucn III mRNAexpression was found to be limited mainly to a few discrete regions ofthe ventral forebrain (FIG. 5). One was in the median preoptic nucleus,where a continuous band of positively labeled cells comprised aninverted Y-shaped midline grouping. Neither of the circumventricularcell groups with which the median preoptic nucleus is intimatelyassociated (the vascular organ of the lamina terminalis and thesubfornical organ) contained positive hybridization signals, and theonly additional sites of Ucn III mRNA expression at this level were overscattered cells in medial and lateral preoptic areas. A second majorlocus of Ucn III mRNA expression appeared as a longitudinally organizedcluster of labeled cells associated with (and essentially encircling)the columns of the fornix throughout the rostral hypothalamus. Thiscluster includes cells situated within the posterior part of the bednucleus of the stria terminalis, anterior and lateral hypothalamic areasand the ill-defined region just lateral to (but seldom within) theparaventricular nucleus of the hypothalamus. The caudal extension ofthis grouping occupied an analagous position, mainly dorsal and lateralto the rostral aspects of the dorsomedial hypothalamic nucleus. Apartfrom this “rostral perifornical” group, scattered positively hybridizedneurons were seen reliably in the ventral part of the anteriorperiventricular nucleus and the retrochiasmatic area. The third majorsite of Ucn III mRNA expression was over a subset of cells in theanterodorsal part of the medial amygdaloid nucleus. In the brain stem,the only reliable site of Ucn III expression was localized discretely toan auditory-related cell group, the superior paraolivary nucleus (FIG.5).

EXAMPLE 12 Acylation of Urocortin III

[0092] The N-termini of the urocortin III proteins of the instantinvention may be extended with acylating agents derived from any of alarge number of carboxyl- and sulfonyl-containing moieties includingadditional amino acids or peptides selected from the group consisting ofD-tyrosine, L-tyrosine, D-tyrosine-glycine, and L-tyrosine-glycine. TheN-termini of urocortin III protein may also be extended with toxins orisocyanates. Addition of acylating agents may increase resistance todegradation (chemical and enzymatic); modulate the solubility of theprotein to allow slow release of therapeutic forms of the proteins; andallowing selective labeling (radioactive, fluorescent, chelators,toxins, photoaffinity, immunospecific, etc).

EXAMPLE 13 Urocortin III Analogs

[0093] Previous studies with ligands for other CRF receptors have shownthat a number of amino acid substitutions can be made to these ligandswithout the ligands losing either the ability to bind to appropriatereceptors or their bioactivity. A number of previous studies withurocortins have shown that one, two or even three substitutions aretolerated easily. In some instances modifications to urocortin resultedin protein with more desirable pharmacological properties. Sinceurocortin III is a small protein, such modification can be most easilyincorporated by peptide synthesis methods well known to those of skillin the art. These include solid phase techniques, partial solid phase,fragment condensation, and classical solution addition. These methodsare especially preferred if nonstandard amino acids are to beincorporated into urocortin III. Alternatively, if the modificationsconsist entirely of natural amino acids, recombinant DNA techniques canbe used for mutagenesis and subsequent expression of modified urocortinIII.

[0094] Mature urocortin III lacks a tyrosine residue. Since tyrosineresidues are useful for the radioiodination of proteins, one possiblemodification of urocortin III would be to substitute tyrosine foranother amino acid in the protein. In previous examples, the addition ofa sequence consisting of Tyr-Gly to the N-terminal end of urocortin wasdescribed. The resulting protein retains CRF receptor binding andbioactivity but would be useful in the radioiodination of the protein.

[0095] Deletion of the first five to eight residues of urocortin wasfound to result in the formation of effective urocortin antagonists.These proteins were capable of binding to CRFreceptors but did notsignificantly stimulate or activate the receptors. It is expected thatdeletion of four to eight amino acids from urocortin III would result ineffective antagonists as well. It may also be possible to createantagonists of CRF-binding proteins from other urocortin III fragments.These antagonists can be effective in elevating levels of the endogenouspeptides, which are normally cleared by CRF-binding protein. Byassociating with the CRF-binding protein and blocking CRF, urocortin,urocortin II, and urocortin III binding to the same protein, theeffective in vivo concentrations of endogenous CRF, Urocortin, UrocortinII and Urocortin III are increased. Such antagonists can beco-administered with other agonists or antagonists of CRF, Urocortin,Urocortin II or Urocortin III for enhancement of the effects thereof.

[0096]FIG. 2C shows the results of a homology comparison betweenurocortin III and equivalent segments of pufferfish urocortin relatedpeptide, mouse urocortin II, human urocortin related peptide, raturocortin, rat corticotropin releasing factor, ovine corticotropinreleasing factor and frog sauvagine. The homology ranges from 20% to77%. An analysis of FIG. 2C reveals amino acids conserved in otherurocortins and urocortin related proteins. While many of the amino acidresidues that are conserved in other urocortins and urocortin relatedproteins are conserved in urocortin III, other amino acid residuesdiffer from those conserved in these other proteins. Therefore,substituting these divergent amino acid residues with those shared bythe other urocortin related proteins provides a means by which to designurocortin III analogs for use as agonists and antagonists. For example,while a lysine residue is present at position 20 of urocortin, andarginine residue is present at the analogous position in rat urocortin,mouse urocortin II, human urocortin related peptide and ratcorticotropin releasing factor. Therefore, a substitution of Lys₂₀ ofurocortin with an arginine residue should produce an urocortin IIIanalog which still associates with the CRF receptors but with a modifiedbinding affinity. Other suggested substitutions in urocortin III includeIle₃, Ile₅, Leu₇, Thr₈, Ile₉, Phe₉, Gly₁₀, His₁₀, Leu₁₁, Leu₁₂, Arg₁₃,Gln₁₃, Leu₁₆, Glu₁₇, Asp₁₇, Arg₂₀, Arg₃₂, Ile₃₄, Leu₃₅, Asp₃₆, Glu₃₆ andVal₃₈. Since many of these substitutions result in an urocortin IIIprotein closer to the consensus sequence of all urocortin relatedproteins, it is expected that many of these will bind more strongly tothe CRF receptors and thus form potent CRF receptor agonists.Alternatively, by the N-terminal deletion of the first four to eightamino acids as described above, these agonists can be converted into CRFreceptor antagonists.

[0097] Other possible substitutions include the replacement of themethionine residues at positions 12 and 35 and/or the leucine residuesat positions 3, 5, 14, 15, 24 and 34 with Nle or C_(α)Me-Leu,₁₅residues. Since comparison of the urocortin III sequence to otherurocortin positions suggests the substitution of leucine at positions 7,11, 12, 16 and 35 or urocortin III, Nle or C_(α)Me-Leu₁₅ may besubstituted in place of Leu at these positions.

[0098] An urocortin III analog containing one or more of the alterationsdescribed above is synthesized. Testing in accordance with the generalprocedure set forth in Example 5 shows improved binding to the CRF-R1,CRF-R2α and/or CRF-R2β receptors by competitive displacement of¹²⁵I[Tyr⁰,Glu¹,Nle¹⁷]sauvagine from Chinese hamster ovary cells stablyexpressing each receptor type and alters intracellular cAMP levels.Further testing indicates that the urocortin III analog has beneficialeffects in the treatment of high body temperature, appetite dysfunction,congestive heart failure, vascular disease and other cardiovascularconditions, gastrointestinal dysfunction, stress and anxiety, migraineheadaches and as a potent inhibitor of angiogenesis.

EXAMPLE 14 Pharmaceutical Administration of Urocortin III and itsAnalogs

[0099] Urocortin III, its analogs or the nontoxic addition saltsthereof, combined with a pharmaceutically or veterinarily acceptablecarrier to form a pharmaceutical composition, may be administered tomammals, including humans, and other animals either intravenously,subcutaneously, intramuscularly, percutaneously, e.g. intranasally,intrapulmonary, intracerebrospinally, sublingually or orally. Thepeptides should be at least about 90% pure and preferably should have apurity of at least about 97%; however, lower purities are effective andmay well be used with animals other than humans. This purity means thatthe intended peptide constitutes the stated weight % of all likepeptides and peptide fragments present.

[0100] Administration of urocortin III or urocortin III agonists tohumans may be employed by a physician to treat high body temperature,appetite dysfunction, congestive heart failure, vascular disease,gastrointestinal dysfunction, stress and anxiety. Urocortin IIIantagonists may be administered to treat congestive heart failure,vascular disease, gastrointestinal dysfunction and migraine headaches orto inhibit angiogenesis. The required dosage will vary with theparticular condition being treated, with the severity of the conditionand with the duration of desired treatment. These peptides may also beused to evaluate hypothalamic pituitary adrenal function in animals withsuspected endocrine or central nervous system pathology by suitableadministration followed by monitoring body functions.

[0101] Such peptides are often administered in the form ofpharmaceutically or veterinarily acceptable nontoxic salts, such as acidaddition salts or metal complexes, e.g., with zinc, iron, calcium,barium, magnesium, aluminum or the like (which are considered asaddition salts for purposes of this application). Illustrative of suchacid addition salts are hydrochloride, hydrobromide, sulphate,phosphate, tannate, oxalate, fumarate, gluconate, alginate, maleate,acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate andthe like. If the active ingredient is to be administered in tablet form,the tablet may contain a binder, such as tragacanth, corn starch orgelatin; a disintegrating agent, such as alginic acid; and a lubricant,such as magnesium stearate. If administration in liquid form is desired,sweetening and/or flavoring may be used, and intravenous administrationin isotonic saline, phosphate buffer solutions or the like may beeffected.

[0102] The peptides should be administered under the guidance of aphysician, and pharmaceutical compositions will usually contain thepeptide in conjunction with a conventional, pharmaceutically orveterinarily-acceptable carrier. Usually, the dosage will be from about1 to about 200 micrograms of the peptide per kilogram of the body weightof the host animal.

EXAMPLE 15 Ucn III and Pancreatic Function

[0103] Examination of the expression of Ucn III mRNA in the rodentsshowed that Ucn III is expressed both in the central nervous system andin the periphery. In the periphery, the pancreas and gastrointestinaltract are major organs of Ucn III expression. Further histologicalstudies showed that within the pancreas, Ucn III is co-localized withinsulin and, therefore, is in the β cells of the Islets of Langerhans.

[0104] To determine the function for this new beta cell product, Ucn IIIgiven intravenously to rats rapidly stimulated glucagon secretion andsubsequently elevates blood sugar levels. It has been shown by othersthat glucagon can act directly on the beta cell to stimulate insulinsecretion in a paracrine manner. Thus, Ucn III may provide a means forthe beta cell to signal the alpha cell, which produces glucagon whichwould then, in turn, drive the beta cell. Thus, Ucn III (or any CRF-R2agonist) may be used to stimulate and sustain islet functions.

[0105] Because insulin and glucagon have opposite effects on blood sugarlevels acutely, it is possible that Ucn III might have long termsalutary effects on islet functions. On the other hand, given thepossible role of intra-islet Ucn III, it is feasible that a CRF-R2antagonist might also be useful to reduce production of glucagon, whichis a major hyperglycemic factor known to exacerbate diabetic glucosecontrol.

[0106] Discussion

[0107] Ucn III was identified by sequence homology screening of thehuman Genbank database. Analyses of the peptide or nucleotide sequencesof the complete proteins or putative mature peptide regions of Ucn IIIand Ucn II suggest that they represent a separate branch of the CRFfamily more closely related to one another than they are to othermembers (FIG. 2D). With 76% identity within the putative mature peptidedomain, Ucn III and puffer fish URP are likely to be orthologous. In theabsence of the identification of any other new urocortin-relatedpeptides, it is likely that the human urocortin-related peptide gene,which is most related to mouse Ucn II, is the human Ucn II ortholog.This suggests that the human URP gene should be renamed human Ucn II.The fact that human Ucn II lacks consensus proteolytic processingresidues at the putative C-terminal region (although the potential amidedonating glycine is present) raises the possibility that the human UcnII prohormone may not yield a peptide similar in size to other membersof the family. The orthologous relationship between murine and human UcnII notwithstanding, the chemical nature and functions of urocortin II inthe human remain at issue

[0108] Both mouse and human Ucn III are highly selective for the type 2CRF receptors and, like Ucn II, exhibit low affinities for type 1receptors and minimal abilities to induce cyclic AMP production in cellsexpressing either endogenous (anterior pituitary corticotropes; FIG. 3B)or transfected (Table 2) CRF-R1. Human Ucn III has lower affinity forthe type 2 receptors than do either mouse Ucn III or human or mouse UcnII. Only four residues differ between human and mouse Ucn III providingstructure/function insight regarding the requirements for high affinityCRF-R2 binding. Human Ucn III is also less potent on human CRF-R2α (datanot shown), indicating that the affinity differences between mouse andhuman Ucn III are not related to the species source of the receptor.However, in spite of its relatively low binding affinity for CRF-R2,human Ucn III is functionally quite potent (EC50<1 nM) to promote cyclicAMP production by cells expressing this receptor. Therefore, both humanand mouse Ucn III exhibit sufficient potency to serve as native ligandsfor CRF-R2. Neither of the CRF-R2 selective ligands, Ucn III or Ucn II,are bound by CRF-BP with high affinity. By contrast, the two ligandswith high affinity for CRF-R1 also have high affinity for CRF-BP.

[0109] From the RNase protection analyses, it is feasible that urocortinIII could gain access to receptors derived from CRF-R2 in both the brainand periphery. In the periphery, Ucn III mRNA is found in the smallintestine and skin, although the cell types in which Ucn III mRNA isexpressed remain to be determined. In the GI tract, CRF-R2 has beenshown to be involved in modulating gut motility (31).

[0110] In the brain, Ucn III mRNA is found in discrete subcorticalregions where its distribution is distinct from that of CRF (32), Ucn(33) and Ucn II (21). While identification of the contexts in which UcnIII may operate must await the results of detailed immunohistochemicaland functional analyses, some initial insights may be gleaned from itsmajor sites of expression in the median preoptic nucleus, termed here asthe rostral perifornical region, and the medial nucleus of the amygdala.By virtue of receiving inputs from both circumventricular structures ofthe lamina terminalis and the brain stem, the median preoptic nucleus isconsidered a key site for the integration of neural and humoral signalsfrom the viscera, related to fluid and cardiovascular homeostasis (34)(35). Among the major targets of its projections are multiple relevantneurosecretory and pre-autonomic populations of the paraventricularand/or supraoptic nuclei of the hypothalamus (36), at least some ofwhich are known to express CRF-R2 (37).

[0111] It is decidedly more difficult to assign potential functions tothe rostral perifornical group of Ucn III-expressing cells, as thiscluster spans several cytoarchitectonically defined cell groups, and isdistinct from the perifornical hypothalamic nucleus recognized by someauthors (38). It may be pointed out, however, that the perifornicalregion has been identified as a sensitive site of action for severalneuroactive agents in stimulating ingestive behavior (39-41), and forexcitatory amino acids in eliciting cardiovascular responses (42).Aspects of the perifornical region have been identified as projecting tosuch major sites of CRF-R2 expression as the lateral septal andventromedial hypothalamic nuclei (43-45), and the sources of localinhibitory (GABAergic) projections to stress-related neuroendocrine andautomomic effectors in the paraventricular nucleus includes a rostralperifornical component (46) whose distribution is very similar to thatof Ucn III-expressing neurons.

[0112] With regard to the third major site of Ucn III expressionidentified herein, anatomical and functional studies have indicated thatmedial nucleus of the amygdala projects extensively to the hypothalamus(including the ventromedial nucleus) and other limbic forebrainstructures (47) and is involved in modulation of behaviors (48, 49) andneuroendocrine function (50, 51) related particularly to reproductionand stress. Overall, the central sites of Ucn III expression describedhere are consistent with potential roles for this peptide in modulatingstress-related autonomic, neuroendocrine and behavioral function,perhaps including some previously thought to be a province of othermembers of this peptide family.

[0113] The distributions of each of the three murine urocortins exhibitsome potential for interacting with type 2 CRF receptors and eachexhibits high affinity for these receptors. By contrast, CRF itself ishighly selective for CRF-R1 and has lower affinity for CRF-R2. Under theassumption that the nomenclature for the three urocortin relatedpeptides becomes accepted, it would be reasonable to consider CRF-R2 tobe a urocortin receptor both in function and in name. The “CRF system”now includes ligands with selectivity for each receptor type as well asthe bivalent ligand, urocortin.

[0114] The following references were cited herein:

[0115] 1. Rivier, C. and W. Vale (1983) Nature 305, 325-327.

[0116] 2. Rivier, J., C. Rivier, and W. Vale (1984) in European PeptideSymposium. Djuronaset, Sweden. pp. 104.

[0117] 3. Vale, et al., (1981) Science 213, 1394-1397.

[0118] 4. Koob, G. F. & Heinrichs, S. C. (1999) Brain Res. 848, 141-152.

[0119] 5. Vaughan, et al., (1995) Nature 378, 287-292.

[0120] 6. Lederis, et al., (1982) Proc. West. Pharmacol. Soc. 25,223-227.

[0121] 7. Montecucchi, et al., (1980) Int. J. Pept. Protein Res. 16,191-199.

[0122] 8. Chen, R., et al. (1993) Proc. Natl. Acad. Sci. USA90,8967-8971.

[0123] 9. Perrin et al. (1995) Proc. Natl. Acad. Sci. USA 92, 2969-2973.

[0124] 10. Chen, et al., (1993) in 23rd Annual Meeting of The Societyfor Neuroscience, Washington, DC, pp. 238.

[0125] 11. Vita, et al., (1993) FEBS 335, 1-5.

[0126] 12. Chang, et al., (1993) Neuron 11, 1187-1195.

[0127] 13. Perrin, et al., (1993) Endocrinology 133, 3058-3061.

[0128] 14. Kishimoto, et al., (1995) Proc. Natl. Acad. Sci. USA 92,1108-1112.

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[0130] 16. Van Pett, et al., (2000) J. Comp. Neurol. 428, 191-212.

[0131] 17. Potter, E., et al. (1994) in 76th Annual Meeting of TheEndocrine Society Anaheim, Calif. p. 217.

[0132] 18. Lovenberg, T. W., et al., (1995) Endocrinology 136,3351-3355.

[0133] 19. Rohde, E., et al. (1996) Biochem Pharmacol52(6), 829-33.

[0134] 20. Bale, T. L., et al. (1999) Nat. Genet. 24(4), 410-414.21.

[0135] 21. Reyes, et al., (2001) Proc. Natl. Acad. Sci. USA 98,2843-2848.

[0136] 22. Pedersen, A. G. & Nielsen, H. (1997) Proc. Int. Conf. Intell.Syst. Mol. Biol. 5, 226-233.

[0137] 23. Devi, L. (1991) Fed. Eur. Biochem. Soc. 280, 189-194.

[0138] 24. Miranda, et al., E. (1994) J. Med. Chem. 37, 1450-1459.

[0139] 25. Miller, C. & Rivier, J. (1996) Biopolymers 40, 265-317.

[0140] 26. Sutton, et al., (1995) Endocrinology 136, 1097-1102.

[0141] 27. Kageyama, K., Suda, T. & Vale, W. W. (2001).

[0142] 28. Vale, et al., (1983) in Methods in Enzymology: NeuroendocrinePeptides, ed. Conn, P. M. (Academic Press, New York), Vol. 103, pp.565-577.

[0143] 29. Bilezikjian, et al., (1996) Endocrinology 137, 4277-4284.

[0144] 30. Simmons, D. M., Arriza, J. L. & Swanson, L. W. (1989) JHistotechnol, in press.

[0145] 31. Nozu, T., Martinez, V., Rivier, J. & Taché, Y. (1999) Am. J.Physiol.: Gastrointest. Liver Physiol. 39, G867-G874.

[0146] 32. Swanson, et al., (1983) Neuroendocrinol. 36, 165-186.

[0147] 33. Bittencourt, et al., (1999) J. Comp. Neurol. 415, 285-312.

[0148] 34. Johnson, A. K. & Loewy, A. D. (1990) in Central Regulation ofAutonomic Functions, eds. Loewy, A. D. & Spyer, K. M. (Oxford UniversityPress, New York), pp. 246-267.

[0149] 35. Saper, C. B. & Levisohn, D. (1983) Brain Res. 288, 21-31.

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[0151] 37. Chalmers, et al., (1995) J. Neurosci. 15, 6340-6350.

[0152] 38. Paxinos, G. & Watson, C. (1986) Academic Press, San Diego,Calif.

[0153] 39. Gillard, et al., (1998) J. Neurosci. 18, 2646-2652.

[0154] 40. Stanley, et al., (1993) Brain Res. 604, 304-317.

[0155] 41. Leibowitz, S. F. & Rossakis, C. (1979) Brain Res. 172,101-113.

[0156] 42. Allen, et al., (1993) J. Comp. Neurol. 330, 421-438.

[0157] 43. Kita, H. & Oomura, Y. (1982) Brain Res. Bull. 8, 53-62.

[0158] 44. Onteniente, B., Menetrey, D., Arai, R. & Calas, A. (1989)Cell Tissue Res. 256, 585-592.

[0159] 45. Szeidemann, et al., (1995) J. Comp. Neurol. 358, 573-583.

[0160] 46. Roland, et al., (1993) J. Comp. Neurol. 332, 123-143.

[0161] 47. Canteras, et al., (1995) J. Comp. Neurol. 360, 213-245.

[0162] 48. Newman, S. W. (1999) Ann. N. Y. Acad. Sci. 877, 242-257.

[0163] 49. Rajendren, G. & Moss, R. L. (1993) Brain Res. 617, 81-86.

[0164] 50. Feldman, et al., (1990) Neuroscience 37, 775-779.

[0165] 51. Dayas, et al., (1999) Eur. J. Neurosci. 11, 2312-2322.

[0166] Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

[0167] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

1 17 1 689 DNA Homo sapiens DNA encoding human urocortin III 1aattcggcac gagggggacc gtttccatag agagggaata tcacagccca 50 cttaggaacaatacctggag aagcaggagc cgagaccccg gagcagccac 100 aagttcatgg ggacgtgcatggggccgccc tcctggccct gaagctgcgc 150 cggcctccct gagcgtttcg ctgcggagggaagtccactc tcggggagag 200 atgctgatgc cggtccactt cctgctgctc ctgctgctgctcctgggggg 250 ccccaggaca ggcctccccc acaagttcta caaagccaag cccatcttca300 gctgcctcaa caccgccctg tctgaggctg agaagggcca gtgggaggat 350gcatccctgc tgagcaagag gagcttccac tacctgcgca gcagagacgc 400 ctcttcgggagaggaggagg agggcaaaga gaaaaagact ttccccatct 450 ctggggccag gggtggagccggaggcaccc gttacagata cgtgtcccaa 500 gcacagccca ggggaaagcc acgccaggacacagccaaga gtccccaccg 550 caccaagttc accctgtccc tcgacgtccc caccaacatcatgaacctcc 600 tcttcaacat cgccaaggcc aagaacctgc gtgcccaggc ggccgccaat650 gcccacctga tggcgcaaat tgggaggaag aagtagagg 689 2 161 PRT HomoSapiens Human urocortin III Precursor 2 Met Leu Met Pro Val His Phe LeuLeu Leu Leu Leu Leu Leu Leu 5 10 15 Gly Gly Pro Arg Thr Gly Leu Pro HisLys Phe Tyr Lys Ala Lys 20 25 30 Pro Ile Phe Ser Cys Leu Asn Thr Ala LeuSer Glu Ala Glu Lys 35 40 45 Gly Gln Trp Glu Asp Ala Ser Leu Leu Ser LysArg Ser Phe His 50 55 60 Tyr Leu Arg Ser Arg Asp Ala Ser Ser Gly Glu GluGlu Glu Gly 65 70 75 Lys Glu Lys Lys Thr Phe Pro Ile Ser Gly Ala Arg GlyGly Ala 80 85 90 Gly Gly Thr Arg Tyr Arg Tyr Val Ser Gln Ala Gln Pro ArgGly 95 100 105 Lys Pro Arg Gln Asp Thr Ala Lys Ser Pro His Arg Thr LysPhe 110 115 120 Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn Leu LeuPhe 125 130 135 Asn Ile Ala Lys Ala Lys Asn Leu Arg Ala Gln Ala Ala AlaAsn 140 145 150 Ala His Leu Met Ala Gln Ile Gly Arg Lys Lys 155 160 3 38PRT Homo Sapiens Human urocortin III (hUcn III) 3 Phe Thr Leu Ser LeuAsp Val Pro Thr Asn Ile Met Asn Leu Leu 5 10 15 Phe Asn Ile Ala Lys AlaLys Asn Leu Arg Ala Gln Ala Ala Ala 20 25 30 Asn Ala His Leu Met Ala GlnIle 35 4 164 PRT Mus musculus Mouse urocortin III Precursor 4 Met LeuMet Pro Thr Tyr Phe Leu Leu Pro Leu Leu Leu Leu Leu 5 10 15 Gly Gly ProArg Thr Ser Leu Ser His Lys Phe Tyr Asn Thr Gly 20 25 30 Pro Val Phe SerCys Leu Asn Thr Ala Leu Ser Glu Val Lys Lys 35 40 45 Asn Lys Leu Glu AspVal Pro Leu Leu Ser Lys Lys Ser Phe Gly 50 55 60 His Leu Pro Thr Gln AspPro Ser Gly Glu Glu Asp Asp Asn Gln 65 70 75 Thr His Leu Gln Ile Lys ArgThr Phe Ser Gly Ala Ala Gly Gly 80 85 90 Asn Gly Ala Gly Ser Thr Arg TyrArg Tyr Gln Ser Gln Ala Gln 95 100 105 His Lys Gly Lys Leu Tyr Pro AspLys Pro Lys Ser Asp Arg Gly 110 115 120 Thr Lys Phe Thr Leu Ser Leu AspVal Pro Thr Asn Ile Met Asn 125 130 135 Ile Leu Phe Asn Ile Asp Lys AlaLys Asn Leu Arg Ala Lys Ala 140 145 150 Ala Ala Asn Ala Gln Leu Met AlaGln Ile Gly Lys Lys Lys 155 160 5 38 PRT Mus musculus Mouse urocortinIII (mUcn III) 5 Phe Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn IleLeu 5 10 15 Phe Asn Ile Asp Lys Ala Lys Asn Leu Arg Ala Lys Ala Ala Ala20 25 30 Asn Ala Gln Leu Met Ala Gln Ile 35 6 38 PRT Takifugu rubripesPufferfish Urocortin Related Peptide (pfURP) (AJ25132) 6 Leu Thr Leu SerLeu Asp Val Pro Thr Asn Ile Met Asn Val Leu 5 10 15 Phe Asp Val Ala LysAla Lys Asn Leu Arg Ala Lys Ala Ala Glu 20 25 30 Asn Ala Arg Leu Leu AlaHis Ile 35 7 38 PRT Takifugu rubripes PEPTIDE 29 Pufferfish (AL175143);Xaa is unknown 7 Phe Ala Leu Ser Leu Asp Val Pro Thr Ser Ile Leu Ser ValLeu 5 10 15 Ile Asp Leu Ala Lys Asn Gln Asp Met Arg Ser Lys Ala Xaa Arg20 25 30 Asn Ala Glu Leu Met Ala Arg Ile 35 8 38 PRT Homo sapiens HumanUrocortin-related peptide (hURP), human urocortin II 8 Ile Val Leu SerLeu Asp Val Pro Ile Gly Leu Leu Gln Ile Leu 5 10 15 Leu Glu Gln Ala ArgAla Arg Ala Ala Arg Glu Gln Ala Thr Thr 20 25 30 Asn Ala Arg Ile Leu AlaArg Val 35 9 38 PRT Mus musculus Mouse Urocortin II (mUcn II) 9 Val IleLeu Ser Leu Asp Val Pro Ile Gly Leu Leu Arg Ile Leu 5 10 15 Leu Glu GlnAla Arg Tyr Lys Ala Ala Arg Asn Gln Ala Ala Thr 20 25 30 Asn Ala Gln IleLeu Ala His Val 35 10 41 PRT Homo sapiens Human Corticotropin ReleasingFactor (hCRF) 10 Ser Glu Glu Pro Pro Ile Ser Leu Asp Leu Thr Phe His LeuLeu 5 10 15 Arg Glu Val Leu Glu Met Ala Arg Ala Glu Gln Leu Ala Gln Gln20 25 30 Ala His Ser Asn Arg Lys Leu Met Glu Ile Ile 35 40 11 41 PRTOvis aries Ovine Corticotropin Releasing Factor (oCRF) 11 Ser Gln GluPro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu 5 10 15 Arg Glu Val LeuGlu Met Thr Lys Ala Asp Gln Leu Ala Gln Gln 20 25 30 Ala His Asn Asn ArgLys Leu Leu Asp Ile Ala 35 40 12 38 PRT Takifugu rubripes PEPTIDE 10Pufferfish Urocortin (AL21886); Xaa is unknown 12 Pro Pro Leu Ser IleAsp Leu Thr Phe Xaa Leu Leu Arg Asn Met 5 10 15 Met Gln Arg Ala Glu MetGlu Lys Leu Arg Glu Gln Glu Lys Ile 20 25 30 Asn Arg Glu Ile Leu Glu GlnVal 35 13 40 PRT Unknown Frog Sauvagine (fSvg) 13 Glu Gly Pro Pro IleSer Ile Asp Leu Ser Leu Glu Leu Leu Arg 5 10 15 Lys Met Ile Glu Ile GluLys Gln Glu Lys Glu Lys Gln Gln Ala 20 25 30 Ala Asn Asn Arg Leu Leu LeuAsp Thr Ile 35 40 14 40 PRT Homo sapiens Human Urocortin (hUcn) 14 AspAsn Pro Ser Leu Ser Ile Asp Leu Thr Phe His Leu Leu Arg 5 10 15 Thr LeuLeu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala 20 25 30 Glu Gln AsnArg Ile Ile Phe Asp Ser Val 35 40 15 40 PRT Mus musculus Mouse Urocortin(mUcn) 15 Asp Asp Pro Pro Leu Ser Ile Asp Leu Thr Phe His Leu Leu Arg 510 15 Thr Leu Leu Glu Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala 20 2530 Glu Gln Asn Arg Ile Ile Phe Asp Ser Val 35 40 16 27 DNA artificialsequence Nested primer based on partial human EST sequence 16 aagagtccccaccgcaccaa gttcacc 27 17 27 DNA artificial sequence Nested primer basedon partial human EST sequence 17 tccctcgacg tccccaccaa catcatg 27

What is claimed is:
 1. An isolated and purified urocortin III proteinselected from the group consisting of human urocortin III and mouseurocortin III.
 2. The isolated and purified urocortin III protein ofclaim 1, wherein said protein is mouse urocortin III derived from aprecursor peptide of amino acid sequence SEQ ID No:
 4. 3. The isolatedand purified urocortin III protein of claim 1, wherein said protein ismouse urocortin III having amino acid sequence SEQ ID No:
 5. 4. Themouse urocortin III protein of claim 3, wherein the N-terminal end ofsaid protein is modified with acylating agents selected from the groupconsisting of carboxyl-containing moieties, sulfonyl-containing moietiesand isocyanates.
 5. The mouse urocortin protein of claim 3, wherein theN-terminal end of said protein is extended with additional amino acidsor peptides selected from the group consisting of D-tyrosine,L-tyrosine, D-tyrosine-glycine, and L-tyrosine-glycine.
 6. The mouseurocortin III protein of claim 3, wherein the N-terminal end of saidprotein is chemically crosslinked to a toxin molecule.
 7. The mouseurocortin III protein of claim 3, wherein the N-terminal end of saidprotein is extended with additional amino acids or peptides selectedfrom the group consisting of D-iodotyrosine, L-iodotyrosine,D-iodotyrosine-glycine, and L-iodotyrosine-glycine and whereinmethionine residues at positions 12 and 35 are replaced with Nle.
 8. Themouse urocortin III protein of claim 7, wherein the iodotyrosine residueis labeled with an ¹²⁵I radioisotope.
 9. A CRF-R2 antagonist comprisingthe mouse urocortin III protein of claim 3 with an N-terminal deletionselected from the group consisting of the first five amino acids, thefirst six amino acid, the first seven amino acids, and the first eightamino acids.
 10. A pharmaceutical composition comprising the CRF-R2antagonist of claim 9 and a pharmaceutically acceptable carrier.
 11. Amethod of treating a pathophysiological state, comprising the step ofadministering the pharmaceutical composition of claim 10 to anindividual in need of such treatment.
 12. The method of claim 11 whereinsaid pathophysiological state is selected from the group consisting ofcongestive heart failure, vascular disease, gastrointestinaldysfunction, diabetes mellitus and migraine headaches.
 13. A method ofinhibiting angiogenesis, comprising the step of administering thepharmaceutical composition of claim 10 to an individual in need of suchtreatment.
 14. The CRF-R2 antagonist of claim 9 wherein the N-terminalend of said antagonist is blocked with an acylating agent selected fromthe group consisting of carboxyl-containing moieties,sulfonyl-containing moieties and isocyanates.
 15. A pharmaceuticalcomposition comprising the CRF-R2 antagonist of claim 14 and apharmaceutically acceptable carrier.
 16. A method of treating apathophysiological state, comprising the step of administering thepharmaceutical composition of claim 15 to an individual in need of suchtreatment.
 17. The method of claim 16 wherein said pathophysiologicalstate is selected from the group consisting of congestive heart failure,vascular disease, gastrointestinal dysfunction, diabetes mellitus andmigraine headaches.
 18. A method of inhibiting angiogenesis, comprisingthe step of administering the pharmaceutical composition of claim 15 toan individual in need of such treatment.
 19. A synthetic urocortin IIIanalog comprising the mouse urocortin III protein of claim 3, whereinsaid protein contains one or more amino acid substitutions selected fromthe group consisting of Ile₃, Nle₃, C_(α)Me-LeU₃, Ile₅, Nle₅,C_(α)Me-Leu₅, Leu₇, Nle₇, Thr₈, Ile₉, Phe₉, Gly₁₀, His₁₀, Leu₁₁, Nle₁₁,Leu₁₂, Nle₁₂, Arg₁₃, Gln₁₃, Nle₁₄, C_(α)Me-Leu₁₄, Nle₁₅, C_(α)Me-Leu₁₅,C_(α)Me-Leu₁₆, Leu₁₆, Nle₁₆, Glu₁₇, Asp₁₇, Nle₁₈, Leu₁₈, Arg₂₀, Nle₂₄,C_(α)Me-Leu₂₄, Arg₃₂, Ile₃₄, Nle₃₄, C_(α)Me-Leu₃₄, Leu₃₅, Nle₃₅, Asp₃₆,Glu₃₆ and Val₃₈.
 20. The synthetic urocortin III analog of claim 19,wherein a methionine residue at position 12 is replaced with Nle. 21.The synthetic urocortin III analog of claim 19, wherein a methionineresidue at position 35 is replaced with Nle.
 22. A pharmaceuticalcomposition comprising the synthetic urocortin III analog of claim 19and a pharmaceutically acceptable carrier.
 23. A method of treating apathophysiological state, comprising the step of administering thepharmaceutical composition of claim 22 to an individual in need of suchtreatment.
 24. The method of claim 23, wherein said pathophysiologicalstate is selected from the group consisting of high body temperature,appetite dysfunction, congestive heart failure, vascular disease,gastrointestinal dysfunction, stress, undesirably low levels of glucagonsecretion or activity and anxiety.
 25. A CRF-R2 antagonist comprisingthe synthetic urocortin III analog of claim 19 with an N-terminaldeletion selected from the group consisting of the first five aminoacids, the first six amino acid, the first seven amino acids, and thefirst eight amino acids.
 26. A pharmaceutical composition comprising theCRF-R2 antagonist of claim 25 and a pharmaceutically acceptable carrier.27. A method of treating a pathophysiological state, comprising the stepof administering the pharmaceutical composition of claim 26 to anindividual in need of such treatment.
 28. The method of claim 27 whereinsaid pathophysiological state is selected from the group consisting ofcongestive heart failure, vascular disease, gastrointestinaldysfunction, diabetes mellitus and migraine headaches.
 29. A method ofinhibiting angiogenesis, comprising the step of administering thepharmaceutical composition of claim 26 to an individual in need of suchtreatment.
 30. The CRF-R2 antagonist of claim 25 wherein the N-terminalend of said antagonist is blocked with an acylating agent selected fromthe group consisting of carboxyl-containing moieties,sulfonyl-containing moieties and isocyanates.
 31. A pharmaceuticalcomposition comprising the CRF-R2 antagonist of claim 29 and apharmaceutically acceptable carrier.
 32. A method of treating apathophysiological state, comprising the step of administering thepharmaceutical composition of claim 31 to an individual in need of suchtreatment.
 33. The method of claim 32 wherein said pathophysiologicalstate is selected from the group consisting of congestive heart failure,vascular disease, gastrointestinal dysfunction, diabetes mellitus andmigraine headaches.
 34. A method of inhibiting angiogenesis, comprisingthe step of administering the pharmaceutical composition of claim 31 toan individual in need of such treatment.
 35. The isolated and purifiedurocortin III protein of claim 1, wherein said protein is humanurocortin III derived from a precursor peptide of amino acid sequenceSEQ ID No:
 2. 36. The isolated and purified urocortin III protein ofclaim 35, wherein said protein is human urocortin III having amino acidsequence SEQ ID No:
 3. 37. The human urocortin III protein of claim 36containing at least one amino acid substitution selected from the groupconsisting of Ile₁₄, Asp₁₉, Lys₂₇, and Gln₃₃ or a combination thereof.38. The human urocortin III protein of claim 37, wherein said proteincontains a single amino acid substitution consisting of Ile₁₄.
 39. Thehuman urocortin III protein of claim 36, wherein the N-terminus of saidprotein is extended with an acylating agent selected from the groupconsisting of carboxyl-containing moieties, sulfonyl-containing moietiesand isocyanates.
 40. The human urocortin III protein of claim 36,wherein the N-terminal end of said protein is extended with additionalamino acids or peptides selected from the group consisting ofD-tyrosine, L-tyrosine, D-tyrosine-glycine, and L-tyrosine-glycine. 41.The human urocortin III protein of claim 36, wherein the N-terminal endof said protein is chemically crosslinked to a toxin molecule.
 42. Thehuman urocortin III protein of claim 36, wherein the N-terminal end ofsaid protein is extended with additional amino acids or peptidesselected from the group consisting of D-iodotyrosine, L-iodotyrosine,D-iodotyrosine-glycine, and L-iodotyrosine-glycine and whereinmethionine residues at positions 12 and 35 are replaced with Nle. 43.The human urocortin III protein of claim 41, wherein the iodotyrosineresidue is labeled with an ¹²⁵ I radioisotope.
 44. A CRF-R2 antagonistcomprising the human urocortin III protein of claim 36 with anN-terminal deletion selected from the group consisting of the first fiveamino acids, the first six amino acid, the first seven amino acids, andthe first eight amino acids.
 45. A pharmaceutical composition comprisingthe CRF-R2 antagonist of claim 44 and a pharmaceutically acceptablecarrier.
 46. A method of treating a pathophysiological state, comprisingthe step of administering the pharmaceutical composition of claim 45 toan individual in need of such treatment.
 47. The method of claim 46wherein said pathophysiological state is selected from the groupconsisting of congestive heart failure, vascular disease,gastrointestinal dysfunction, diabetes mellitus and migraine headaches.48. A method of inhibiting angiogenesis, comprising the step ofadministering the pharmaceutical composition of claim 45 to anindividual in need of such treatment.
 49. A CRFR2 antagonist comprisingthe CRF-R2 antagonist of claim 44 wherein the N-terminal end of saidantagonist is blocked with an acylating agent selected from the groupconsisting carboxyl-containing moieties, sulfonyl-containing moietiesand isocyanates.
 50. A pharmaceutical composition comprising the CRF-R2antagonist of claim 49 and a pharmaceutically acceptable carrier.
 51. Amethod of treating a pathophysiological state, comprising the step ofadministering the pharmaceutical composition of claim 50 to anindividual in need of such treatment.
 52. The method of claim 51 whereinsaid pathophysiological state is selected from the group consisting ofcongestive heart failure, vascular disease, gastrointestinaldysfunction, diabetes mellitus and migraine headaches.
 53. A method ofinhibiting angiogenesis, comprising the step of administering thepharmaceutical composition of claim 50 to an individual in need of suchtreatment.
 54. A synthetic urocortin III analog comprising the humanurocortin III protein of claim 36, wherein said protein contains one ormore amino acid substitutions selected from the group consisting ofIle₃, Nle₃, C_(α)Me-Leu₃, Ile₅, Nle₅, C_(α)Me-Leu₅, Leu₇, Nle₇, Thr₈,Ile₉, Phe₉, Gly₁₀, His₁₀, Leu₁₁, Nle₁₁, Leu₁₂, Nle₁₂, Arg₁₃, Gln₁₃,Nle₁₄, C_(α)Me-Leu₁₄, Nle₁₅, C_(α)Me-Leu₁₅, C_(α)Me-Leu₁₆, Leu₁₆, Nle₁₆,Glu₁₇, Asp₁₇, Nle18, Leu18, Arg₂₀, Nle₂₄, C_(α)Me-Leu₂₄, Arg₃₂, Ile₃₄,Nle₃₄, C_(α)Me-Leu₃₄, Leu₃₅, Nle₃₅, Asp₃₆, Glu₃₆, and Val₃₈.
 55. Theurocortin III analog of claim 54, wherein a methionine residue atposition 12 is replaced with Nle.
 56. The urocortin III analog of claim54, wherein a methionine residue at position 35 is replaced with Nle.57. A pharmaceutical composition comprising the urocortin III analog ofclaim 54 and a pharmaceutically acceptable carrier.
 58. A method oftreating a pathophysiological state, comprising the step ofadministering the pharmaceutical composition of claim 57 to anindividual in need of such treatment.
 59. The method of claim 58,wherein said pathophysiological state is selected from the groupconsisting of high body temperature, appetite dysfunction, congestiveheart failure, vascular disease, gastrointestinal dysfunction, stress,undesirably low levels of glucagon secretion or activity and anxiety.60. A CRF-R2 antagonist comprising the human urocortin III analog ofclaim 54 with an N-terminal deletion selected from the group consistingof the first five amino acids, the first six amino acid, the first sevenamino acids, and the first eight amino acids.
 61. A pharmaceuticalcomposition comprising the CRF-R2 antagonist of claim 60 and apharmaceutically acceptable carrier.
 62. A method of treating apathophysiological state, comprising the step of administering thepharmaceutical composition of claim 61 to an individual in need of suchtreatment.
 63. The method of claim 62 wherein said pathophysiologicalstate is selected from the group consisting of congestive heart failure,vascular disease, gastrointestinal dysfunction, diabetes mellitus andmigraine headaches.
 64. A method of inhibiting angiogenesis, comprisingthe step of administering the pharmaceutical composition of claim 61 toan individual in need of such treatment.
 65. The CRF-R2 antagonist ofclaim 60 wherein the N-terminal end of said antagonist is blocked withan acylating agent selected from the group consisting ofcarboxyl-containing moieties, sulfonyl-containing moieties andisocyanates.
 66. A pharmaceutical composition comprising the CRF-R2antagonist of claim 65 and a pharmaceutically acceptable carrier.
 67. Amethod of treating a pathophysiological state, comprising the step ofadministering the pharmaceutical composition of claim 66 to anindividual in need of such treatment.
 68. The method of claim 67 whereinsaid pathophysiological state is selected from the group consisting ofcongestive heart failure, vascular disease, gastrointestinaldysfunction, diabetes mellitus and migraine headaches.
 69. A method ofinhibiting angiogenesis, comprising the step of administering thepharmaceutical composition of claim 66 to an individual in need of suchtreatment.
 70. The urocortin III protein of claim 1, wherein saidprotein has been modified to contain a molecular label.
 71. The proteinof claim 70, wherein said label is selected from the group consisting offluorescent labels, photoaffinity labels and radioactive markers.
 72. Amethod of determining whether a cell has urocortin III receptors,comprising the steps of: incubating said cells with the urocortin IIIprotein of claim 70, detecting said molecular label, wherein thepresence of said label on said cells indicates that said cells haveurocortin III receptors.
 73. A conjugate of the protein of claim 1linked to a toxin.
 74. An antibody directed against the urocortin IIIprotein of claim
 1. 75. The antibody of claim 74, wherein said antibodyis a monoclonal antibody.
 76. The antibody of claim 74 conjugated to amolecular label.
 77. The antibody of claim 76, wherein said label isselected from the group consisting of fluorescent labels, photoaffinitylabels and radioactive markers.